Project 1: Summary/Abstract Pancreatitis is a potentially fatal disease with significant morbidity and mortality, the pathogenesis of which remains obscure and for which no specific or effective treatment exists. Our findings indicate that lysosomal function is defective in pancreatitis, leading to impaired autophagy and key pathologic responses of pancreatitis such as the accumulation of active trypsin and vacuoles in acinar cells. The mechanisms of lysosomal/autophagic dysfunction in pancreatitis are not known. Rab proteins are small GTPases the principal function of which is to oversee vectorial transport of proteins and membranes between organelles. They coordinate all aspects of the endo-lysosomal system, and also regulate autophagy. Rab9, in particular, regulates trafficking of mannose-6-phosphate receptors (M6PRs) that control the delivery of newly synthesized hydrolases to the lysosome. Rabs are synthesized as soluble proteins and then undergo lipidation (prenylation), which is mediated by Rab escort protein (REP) and is necessary for Rab delivery to membranes and subsequent activation. Neither the effect of pancreatitis on Rab GTPases nor their roles in the pathogenesis of this disease have been investigated. Our preliminary results indicate that acinar cell Rab functioning is disordered in pancreatitis due to their decreased delivery to membranes caused by a decrease in REP1. In this application, we propose a novel concept for the mechanism of pancreatitis, namely, that global Rab dysfunction is a critical pathogenic event initiating pancreatitis. We hypothesize the following chain of events: REP1 decrease causes deficient Rab prenylation, resulting in a decrease in membrane-bound Rabs. The decrease in membrane Rab9 leads to decreased M6PR levels and ultimately, to the lysosomal/autophagic dysfunction. Thus, Rab dysfunction underlies pancreatitis pathologies. The hypothesis will be tested in three Specific Aims: 1). Investigate Rab dysfunction in pancreatitis and the role of Rab prenylation as the underlying mechanism. 2). Determine the roles of Rab prenylation and Rab9-M6PR pathway in the lysosomal/autophagic dysfunction in pancreatitis. 3). Determine the role of Rab dysfunction in key pancreatitis responses: trypsinogen activation, inflammation and acinar cell death. The proposal will use genetically modified mouse models, human and mouse acinar cells, and various biochemical and cell biological techniques to examine the mechanisms of Rab dysfunction in pancreatitis. Elucidation of these mechanisms will lead to identification of novel molecular targets, such as REP1 and Rab9, for therapeutic interventions to restore lysosomal/autophagic function and thus prevent or ameliorate pancreatitis. Importantly, similar pathogenic mechanisms are likely to operate in other disease states as well.